P458: HIPSC DISEASE MODELING TO STUDY LEUKEMIA DEVELOPMENT IN HAX1 VS ELANE ASSOCIATED CONGENITAL NEUTROPENIA
Authors: Benjamin Dannenmann, Maksim Klimiankou, Cornelia Zeidler, Karl Welte, Julia Skokowa
Journal: HemaSphere (2023)

Background: Several inherited mutations in HAX1 and ELANE cause severe congenital neutropenia (CN). CN is a pre-leukemic bone marrow failure syndrome with a high risk of developing MDS/AML associated with the co-acquisition of CSF3R and RUNX1 mutations (Skokowa et al. Blood. 2014). How genetic lesions in CSF3R and RUNX1 contribute to progression to AML/MDS in CN is still poorly understood. Aims: We aimed to compare intracellular signaling pathways responsible for leukemogenesis in HAX1 vs ELANE-CN. We further aimed to compare HAX1 and ELANE-CN/AML phenotypes during hematopoietic differentiation of iPSCs and the role of missense vs non-sense RUNX1 mutations in leukemogenesis. Lastly, we planned to identify small molecules targeting major pathways involved in leukemia development in CN. Methods: We established a model for step-wise leukemia progression in HAX1- and ELANE - CN by lentiviral reprogramming of HAX1 and ELANE - CN CD34+ cells at various stages of leukemogenesis in combination with CRISPR/Cas9-mediated gene editing of iPSCs, followed by EB-based hematopoietic differentiation of iPSCs. iPSC-derived CD34+CD45+cells were tested for differentiation capacity by CFU-Assay and analyzed by RNA-sequencing. Results:HAX1- and ELANE-CN/AML-iPSC-derived CD34+CD45+ HSPCs showed reduced granulocytic differentiation in CFU-Assay and increased proliferation rate. AML phenotype was comparable for non-sense and missense RUNX1 mutations in HAX1-CN/AML. RNA-seq analysis of iPSC-derived CD34+CD45+ cells showed high similarity between ELANE- and HAX1-CN/AML revealing features of RUNX1 mutant leukemia, upregulation of inflammatory response and MYC targets. We also identified GATA1/2 transcription factors, as well as HIPK2 and MAPK1,3,14 kinases as main regulators of DEGs. BAALC upregulation was identified as a key leukemogenic event and BAALC KO restored granulocytic differentiation (Dannenmann et al. Cell Stem Cell. 2021). Deregulated signaling pathways in both HAX1 and ELANE-CN/AML were BAALC-dependent. Connectivity Map analysis of RNA-seq data revealed that MAPK14 (p38) pathway inhibition by small molecule CMPD1, a MK2a inhibitor, correlated with BAALC KO phenotype in CN/AML. We showed that MK2a is hyperphosphorylated in CN/AML in a BAALC-dependent manner. CMPD1 treatment was able to selectively eliminate CN/AML-iPSC-derived blasts, primary CN/AML blasts with both non-sense and missense RUNX1 mutations, de novo AML blasts with high BAALC expression and also transplanted CN/AML-blasts in vivo in zebrafish embryos. CMPD1 treatment was effective for both HAX1- and ELANE-CN/AML blasts. Summary/Conclusion: We established an iPSC model of leukemic development in HAX1 and ELANE associated congenital neutropenia displaying reduced granulocytic differentiation and increased proliferation rate for both HAX1 and ELANE-CN/AML iPSC-derived HSPCs. BAALC is a key oncogene in congenital neutropenia. BAALC KO or MK2a inhibition induces differentiation and inhibits proliferation of BAALChigh AML blasts in HAX1 and ELANE-CN/AML. BAALC exerts its leukemogenic effects via MAPK14 (p38)/MK2a pathway. MK2a inhibition by CMPD1 might be a possible treatment strategy for CN/AML and de novo AML with high BAALC expression. Keywords: Severe congenital neutropenia, Acute myeloid leukemia, Leukemogenesis, RUNX1